Introduction

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₁-Adrenergic receptors (₁-ARs) are G-protein-coupled receptors and mediate some of the physiological actions of noradrenaline (NA). Three ₁-AR subtypes have been cloned and they are referred to as _1A-AR, _1B-AR, and _1D-AR (Hieble et al., 1995). Each of these subtypes has a distinct pharmacological profile (Michel et al., 1995) and a distinct tissue distribution of its mRNA (Price et al., 1994a, 1994b). Several studies have demonstrated that ₁-ARs can activate a variety of effectors including phospholipase C, phospholipase D, phospholipase A₂, cAMP metabolism and various ion channels (Davis et al., 1978; Burch et al., 1986; Apkon and Nerbonne, 1988; Wilson and Minneman, 1990; Llahi and Fain, 1992). However, little is known about the potential biological significance of the various specific ₁-AR subtypes being expressed in the same cells. In the present study, we focused on the Ca²⁺ channels activated by the binding of NA to each ₁-AR subtype. A previous report showed that NA evoked a transient increase in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) followed by a sustained increase in [Ca²⁺]_i in Chinese hamster ovary (CHO) cells stably expressing _1A-, _1B-, or _1D-ARs (CHO-_1A, CHO-_1B, or CHO-_1D, respectively) (Horie et al., 1995). However, the Ca²⁺ channels activated by NA in the three cell types were not characterized in that study. Moreover, another report showed that NA failed to induce a sustained increase in [Ca²⁺]_i in CHO-_1B and CHO-_1D (Perez et al., 1993). Therefore, the first purpose of the present study was to identify and compare the Ca²⁺ channels in CHO-_1A, CHO-_1B, and CHO-_1D that are activated by NA using receptor-operated Ca²⁺ channel blockers LOE 908 and SK&F 96365 (Merritt et al., 1990; Encabo et al., 1996) and L-type voltage-operated Ca²⁺ channel (VOCC) blocker nifedipine. We have shown recently that endothelin-1 activates two types of Ca²⁺-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and a store-operated Ca²⁺ channel (SOCC) (Iwamuro et al., 1999). Importantly, we have also shown that these channels can be distinguished by their sensitivity to SK&F 96365 and LOE 908. Thus, NSCC-1 is sensitive to LOE 908 and resistant to SK&F 96365; NSCC-2 is sensitive to both LOE 908 and SK&F 96365; and SOCC is resistant to LOE 908 and sensitive to SK&F 96365 (Iwamuro et al., 1999).

The NA-induced arachidonic acid release in vascular smooth muscle cells depends on Ca²⁺ influx (Nebigil and Malik, 1992). In contrast, the NA-induced arachidonic acid release in CHO cells expressing ₁-ARs is mediated through phospholipase A₂ via a pertussis toxin-sensitive pathway (Perez et al., 1993). Moreover, it was concluded that this response was independent of both Ca²⁺ channel activation and Ca²⁺ mobilization (Perez et al., 1993). However, the Ca²⁺ influx through voltage-independent Ca²⁺ channels was not examined in that report. The second purpose of the present study was to examine whether the influx of extracellular Ca²⁺ through voltage-independent Ca²⁺ channels plays a role in the NA-induced arachidonic acid release using LOE 908.

	Experimental Procedures

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Materials. Human _1A-, _1B-, and _1D-adrenergic receptor cDNA (Takahashi et al., 2000) were kindly provided by Dr. Ikunobu Muramatsu (Fukui Medical University, Japan).

Cell Culture. CHO cells were maintained in F-12 medium supplemented with 10% fetal calf serum (FCS) under a humidified 5% CO₂/95% air atmosphere.

Stable Expression of _1A-, _1B-, or _1D-Adrenergic Receptors in CHO Cells. The procedures for construction and subcloning of receptor cDNAs were as previously described (Sakamoto et al., 1993). In brief, each expression vector that carried the cDNA construct encoding human _1A-AR, _1B-AR, or _1D-AR was cotransfected with pSVbsr^r plasmid into CHO cells by lipofection using LipofectAMINE (Invitrogen, Gaithersburg, MD) according to the manufacturer's instructions. Cell populations expressing the bsr^r gene product were selected in F-12 medium supplemented with 10% FCS and 0.5 µg/ml blasticidine. From these selected populations, clonal cell lines were isolated by colony lifting and were maintained in the same selection medium.

Radioligand Binding Assays. The [³H]prazosin binding assay was performed as described previously (Michel et al., 1993). Briefly, subconfluent transfected cells in the 150-mm plates were washed twice with phosphate-buffered saline (PBS) and harvested by scraping. The harvested cells were suspended in ice-cold assay buffer (50 mM Tris-HCl and 1 mM EDTA, pH 7.4), sonicated, and centrifuged at 3000g (4°C) for 10 min. The supernatant was then centrifuged at 80,000g (4°C) for 30 min, the pellet was resuspended in assay buffer, and this was used in the binding experiments. The protein concentration was measured with the BCA Microprotein Assay Kit (Pierce, Rockford, IL). The membrane preparation (50 µg of protein) was incubated with various concentrations of [³H]prazosin in a total volume of 1 ml at 25°C for 45 min. In competition binding experiments, the membrane preparation was incubated with 200 pM [³H]prazosin and various concentrations of unlabeled drug at 25°C for 45 min. Nonspecific binding was defined as binding in the presence of 10 µM phentolamine. The incubation was terminated by rapid filtration onto Whatman GF/C filters. The filters were washed four times with ice-cold 50 mM Tris-HCl (pH 7.4) and dried. The level of filter-bound radioactivity was determined by liquid scintillation counting.

Monitoring of [Ca²⁺]_i. The monitoring of [Ca²⁺]_i was performed as described previously (Enoki et al., 1995). Briefly, cells were loaded with fluo-3 by incubating the cells with 10 µM fluo-3/AM at 37°C under reduced light for 30 min. After washing, the cells were suspended at a density of approximately 2 × 10⁷ cells/ml, and 0.5-ml aliquots were used for measurement of fluorescence by a CAF 110 spectrophotometer (Jasco, Tokyo, Japan) with an excitation wavelength of 490 nm and an emission wavelength of 540 nm.

Electrophysiology. Cells were perfused with Krebs-HEPES solution and visualized with Nomarski optics (Zeiss, Tokyo, Japan). Whole-cell recordings were made with thin-wall borosilicate glass patch pipettes (resistance, 3-5 M) as previously described (Enoki et al., 1995). The Krebs-HEPES solution contained 140 mM NaCl, 3 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 11 mM glucose, and 10 mM HEPES (adjusted to pH 7.3 with NaOH). The pipettes were filled with Cs-aspartate solution containing 120 mM Cs-aspartate, 20 mM CsCl, 2 mM MgCl₂, 10 mM HEPES, and 10 mM EGTA, adjusted to pH 7.3 with CsOH. Tight-seal whole-cell currents were recorded with an EPC7 patch-clamp amplifier (List, Darmstadt, Germany). The perfusion rate was maintained at 2.2 to 2.5 ml/min, and the bath volume was ~1.0 ml. All experiments were performed under voltage-clamp at a holding potential of 60 mV at room temperature (22-24°C). To test the contribution of the Cl current, the bath solution was switched from Krebs-HEPES to a solution with a low Cl concentration that contained 140 mM sodium gluconate, 3 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 11 mM glucose, and 10 mM HEPES, pH 7.3. The permeability of Ca²⁺ through channels was measured in a Ca²⁺/NMDG solution containing 30 mM CaCl₂, 100 mM NMDG chloride, 11 mM glucose, and 10 mM HEPES, adjusted to pH 7.3 with Tris.

Formation of Inositol Phosphates. The level of formation of inositol phosphates (IPs) was determined as described previously (Sugawara et al., 1996). Briefly, cells in 24-well plates were incubated with myo-[³H]inositol (final concentration, 5 µCi/ml) in 0.3 ml of Ham's F-12 medium supplemented with 10% FCS for 18 h. After washing, the cells were incubated with or without various concentrations of ET-1 for 30 min, and the reaction was terminated by adding ice-cold perchloric acid. After neutralization with KOH and Tris, the samples were applied onto small columns of AG1X8 (100-200 mesh, Cl form; Bio-Rad, Hercules, CA) to separate the total IPs from the myo-[³H]inositol. The [³H]IPs were eluted with 1 N HCl, and the radioactivity was counted with a liquid scintillation counter.

[³H]Arachidonic Acid Release. The level of [³H]arachidonic acid release was determined as described previously (Perez et al., 1993). Briefly, cells in 100-mm dishes were incubated overnight with [³H]arachidonic acid (final concentration, 1 µCi/ml). After washing, the cells were treated with LOE 908 or EGTA for 15 min. Then, 10 nM NA was added, and after 5 min the medium was removed, acidified with 100 µl of 1 N formic acid, and extracted with 3 ml of chloroform. The extracts were evaporated to dryness, resuspended in 50 µl of chloroform, and applied to silica gel plates for thin-layer chromatography (Merck, Darmstadt, Germany). The plates were developed in heptane/diethyl ether/acetic acid/water (v/v; 75:25:4). The distance of movement was visualized with iodine vapor. The plate was scraped, and the radioactivity was counted with a liquid scintillation counter.

Statistical Analysis. All results are expressed as mean ± S.E.M.

	Results

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Stable Expression of _1A-, _1B-, or _1D-ARs in CHO Cells. CHO cells were chosen for expression of ₁-AR subtypes because these cells allow the expression of a transfected gene in a stable manner, and they lack endogenous ₁-ARs (Perez et al., 1993).

Change in the [Ca²⁺]_i in CHO-_1A, CHO-_1B, and CHO-_1D upon Treatment with NA. NA at 10 nM induced a sustained increase in [Ca²⁺]_i in all three cell types (Fig. 1, A, D, and G). On the other hand, at concentrations  100 nM, NA induced a biphasic increase in [Ca²⁺]_i consisting of an initial transient peak and a subsequent sustained increase in all three cell types (Fig. 1, B, E, and H). The receptor specificity was checked using WB-4101, chloroethylclonidine, and BMY7378, which are antagonists of _1A-AR, _1B-AR and _1D-AR, and _1D-AR, respectively (Ipsen et al., 1997; Jarajapu et al., 2001). That is 1) WB-4101 inhibited NA-induced increase in [Ca²⁺]_i in CHO-_1A but not in CHO-_1B or CHO-_1D, 2) chloroethylclonidine inhibited NA-induced increase in [Ca²⁺]_i in CHO-_1B and CHO-_1D but not in CHO-_1A, and 3) BMY7378 inhibited NA-induced increase in [Ca²⁺]_i in CHO-_1D but not in CHO-_1A or CHO-_1B (data not shown). Therefore, NA-induced increase in [Ca²⁺]_i was mediated by ₁-ARs. When the extracellular Ca²⁺ was removed from the bath solution, upon NA treatment, the transient peak was not affected but the sustained increase by either concentration of NA (10 nM or 100 nM) was abolished in all three cell types (Fig. 1, C, F, and I). The magnitude of the transient peak and the magnitude of the sustained increase in [Ca²⁺]_i depended on the concentration of NA (Fig. 2). The maximal values of the transient peak in all three cell types were similar. The maximal values of the sustained increase in [Ca²⁺]_i in all three cell types were almost similar (Fig. 2). The EC₅₀ value of NA (about 5 nM) for the sustained increase seemed to be smaller than that for the transient peak (about 50 nM) (Fig. 2). We used several different CHO-_1A, CHO-_1B, and CHO-_1D clones to examine the degree of the NA-induced sustained increase in [Ca²⁺]_i. Assays on several independent clones with various receptor densities suggested that, within the range of receptor densities that we could obtain, the degree of the NA-induced sustained increase in [Ca²⁺]_i was independent of receptor density (data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Original tracings of the [Ca2+]i in CHO-1A, CHO-1B, and CHO-1D that were treated with various concentrations of NA. CHO-1A (A-C), CHO-1B (D-F), or CHO-1D (G-I) cells were loaded with a Ca2+ indicator, fluo-3, and subjected to monitoring of [Ca2+]i in the presence (A, B, D, E, G, and H) or absence (C, F, and I) of extracellular Ca2+. The cells were exposed to NA at 10 (A, D, and G) or 100 nM (B, C, E, F, H, and I) in the bath solution during the time indicated by the horizontal bar. In case of the monitoring of [Ca2+]i in the absence of extracellular Ca2+, the cells were exposed to Ca2+-free solution containing 3 mM EGTA for 5 min before adding NA.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Concentration-response curves of the magnitude of the transient increase (open circles) and the magnitude of the sustained increase (closed circles) in [Ca2+]i upon treatment of CHO-1A, CHO-1B, and CHO-1D with NA. CHO-1A (A), CHO-1B (B), or CHO-1D (C) cells were loaded with a Ca2+ indicator, fluo-3, and subjected to monitoring of [Ca2+]i. Each point represents the mean ± S.E.M. of five experiments.

Pharmacological Properties of the NA-Induced Increase in [Ca²⁺]_i in CHO-_1A, CHO-_1B, and CHO-_1D. In all three cell types, the sustained increase in [Ca²⁺]_i induced by 100 nM NA was suppressed by LOE 908 in a concentration-dependent manner, and maximal inhibition was observed at concentrations of LOE 908  3 µM (Fig. 3). The inhibition by LOE 908 was virtually complete regardless of the concentration of NA (data not shown). On the other hand, the NA-induced sustained increase in [Ca²⁺]_i was resistant to SK&F 96365 (Fig. 3) or nifedipine (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Original tracings of the [Ca2+]i in CHO-1A (A), CHO-1B (C), and CHO-1D (E) illustrating the effect of SK&F 96365 and LOE 908 after being treated with 100 nM NA. Cells loaded with fluo-3 were stimulated with 100 nM NA. After the [Ca2+]i reached a steady state, the cells were exposed to 10 µM SK&F 96365 and subsequently 3 µM LOE 908 in the bath solution during the times indicated by the respective horizontal bar. Concentration-response curves of the inhibition of the NA-induced increase in [Ca2+]i by LOE 908 (open circles) or SK&F 96365 (closed circles) in CHO-1A (B), CHO-1B (D), or CHO-1D (F). The cells were stimulated with 100 nM NA. After the [Ca2+]i reached a steady state, increasing concentrations of either LOE 908 or SK&F 96365 were added. The [Ca2+]i following stimulation with 100 nM NA was set at 100%, and the [Ca2+]i before stimulation with NA was set at 0%. The [Ca2+]i following the addition of LOE 908 or SK&F 96365 was represented on this scale. Each point represents the mean ± S.E.M. of five experiments.

Characterization of the Currents Induced by NA in CHO-_1A, CHO-_1B, and CHO-_1D with Whole-Cell Recordings of Patch Clamp. To elucidate the ionic channels activated by NA in CHO-_1A, CHO-_1B, and CHO-_1D, whole-cell recordings were performed. Stimulation with 100 nM NA induced an inward current with an increase in baseline "noise" in all three cell types (Fig. 4). The currents induced by NA in CHO-_1A, CHO-_1B, and CHO-_1D showed linear current-voltage relationships, with a reversal potential of 5.4 ± 0.8 mV (n = 10), 4.7 ± 0.3 mV (n = 10), and 3.8 ± 0.4 mV (n = 10), respectively (Fig. 4). The current-voltage relationships induced by NA in CHO-_1A, CHO-_1B, and CHO-_1D were not affected by reducing the concentration of Cl in the bath. In the condition of low extracellular Cl concentration, the reversal potential of the CHO-_1A, CHO-_1B, and CHO-_1D was 3.6 ± 0.2 mV (n = 6), 3.2 ± 0.4 mV (n = 6), and 4.5 ± 0.4 mV (n = 6), respectively. To test whether the channels activated by NA are permeable to Ca²⁺, all cations except Ca²⁺ in the bath solution were replaced with the nonpermeant cation NMDG, whereas the concentration of Ca²⁺ was increased from 1 to 30 mM. Even under such conditions, NA induced an inward current in all three cell types. Under this condition, the reversal potential of the CHO-_1A, CHO-_1B, and CHO-_1D was 10.3 ± 0.6 mV (n = 6), 10.4 ± 0.5 mV (n = 6), and 10.8 ± 0.5 mV (n = 6), respectively.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Whole-cell recordings of the currents in CHO-1A, CHO-1B, or CHO-1D that were incubated with 100 nM NA and then treated with LOE 908. A, C, and E, original tracings illustrating the whole-cell currents in CHO-1A (A), CHO-1B (C), or CHO-1D (E) that had been activated by NA and then treated with LOE 908. The cells were clamped at a holding potential of 60 mV with the whole-cell configuration, and the cells were exposed to 100 nM NA in the bath solution during the time indicated by the horizontal bar. After the NA-induced current had reached a steady state, LOE 908 was added to the bath solution at a final concentration of 3 µM during the time indicated by the respective horizontal bar. B, D, and F, the current-voltage relationships of the currents induced by 100 nM NA and subsequently 3 µM LOE 908 in CHO-1A (B), CHO-1B (D), or CHO-1D (F). At the times indicated by x, y, and z, when the cells were in a stable condition before or after the addition of NA and after the addition of LOE 908, a voltage step of 100 ms in duration was applied. Voltage steps ranging from 100 mV to +80 mV in 20-mV increments were applied. The NA-induced current at each membrane potential was obtained by subtracting the current at x from that at y (open circles), or the current at z from that at y (closed circles), and these were plotted against the membrane potential.

Pharmacological Properties of the Whole-Cell Currents Induced by Stimulation of CHO-_1A, CHO-_1B, and CHO-_1D with NA. In all three cell types, the current induced by 100 nM NA was abolished by 3 µM LOE 908 (Fig. 4), whereas it was resistant to 10 µM SK&F 96365 (data not shown). The currents inhibited by LOE 908 in the three cell types showed a linear current-voltage relationship and a reversal potential of 6 to 2 mV (Fig. 4).

Formation of Inositol Phosphates in CHO-_1A, CHO-_1B, and CHO-_1D after Stimulation with NA. Based on the results of the [Ca²⁺]_i monitoring and a previous report (Gardner, 1989), there is a possibility that IP production is involved in the sustained increase in [Ca²⁺]_i induced by NA. To clarify whether the NA-induced activation of Ca²⁺ channels depends on depletion of the intracellular Ca²⁺ store after IP production and, therefore, whether the activated Ca²⁺ channels are SOCCs, we measured the formation of IPs following stimulation with NA and examined the pharmacological properties of the SOCCs using thapsigargin in CHO-_1A, CHO-_1B, and CHO-_1D.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Formation of IPs in CHO-1A (squares), CHO-1B (triangles), and CHO-1D (circles) following stimulation with various concentrations of NA. Cells that had been incubated with myo-[3H]inositol for 18 h were stimulated with various concentrations of NA for 30 min. The level of total IPs in the cell extract was determined as described under Experimental Procedures. Each point represents the mean ± S.E.M. of five experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   A and B, original tracing of the [Ca2+]i in CHO-1A illustrating the inhibitory effect of SK&F 96365 (A) or LOE 908 (B) on the thapsigargin-induced increase in [Ca2+]i, as an index of the activity of SOCCs. Wild-type CHO cells were loaded with fluo-3 and subjected to monitoring of [Ca2+]i. The cells were exposed to 0.1 µM thapsigargin during the period of time indicated by the horizontal bar. After the [Ca2+]i reached a steady state, either 10 µM SK&F 96365 or 10 µM LOE 908 was added to the bath solution. C, original tracing of the [Ca2+]i illustrating the effect of thapsigargin on increase in [Ca2+]i in CHO-1A preincubated with NA. After the 100 nM NA-induced increase in [Ca2+]i reached a steady state, 0.1 µM thapsigargin was added to the bath solution.

Effect of LOE 908 on NA-Induced Arachidonic Acid Release in CHO-_1A, CHO-_1B, and CHO-_1D. NA at 10 nM caused a 3-fold increase in arachidonic acid release in all three cell types (Fig. 7). This NA-induced increase in arachidonic acid release was nearly completely blocked by chelation of extracellular Ca²⁺ with EGTA (Fig. 7). In addition, 3 µM LOE 908 inhibited the NA-induced increase in arachidonic acid release (Fig. 7). On the other hand, pertussis toxin inhibited the NA-induced increase in arachidonic acid release as described previously (Perez et al., 1993).

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Effect of NA on arachidonic acid release and inhibitory effect of EGTA or LOE 908 on the NA-induced increase in arachidonic acid release in CHO-1A (open bars), CHO-1B (closed bars), or CHO-1D (hatched bars). The levels of arachidonic acid release were determined as described under Experimental Procedures. Cells were incubated for 15 min with or without 5 mM EGTA or 3 µM LOE 908 and then stimulated with 10 nM NA. Each bar represents the mean ± S.E.M. of five experiments.

	Discussion

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There is no consensus as to whether NA activates the influx of extracellular Ca²⁺ influx in CHO-_1A, CHO-_1B, and CHO-_1D (Perez et al., 1993; Horie et al., 1995). Based on the results of the present study, we conclude that NA induces Ca²⁺ influx in CHO-_1A, CHO-_1B, and CHO-_1D (Figs. 1 and 4). Moreover, the magnitude of the transient increase and that of the sustained increase in [Ca²⁺]_i were similar in all three cell types (Figs. 1 and 2). These results differ from the previous observation that the level of the NA-induced sustained increase in [Ca²⁺]_i in CHO-_1D was smaller than that in CHO-_1A or CHO-_1B (Horie et al., 1995). However, this report showed that NA induced sustained increase in [Ca²⁺]_i even in the absence of extracellular Ca²⁺ in CHO-_1A or CHO-_1B (Horie et al., 1995). Therefore, we have doubts about their data on monitoring of NA-induced increase in [Ca²⁺]_i.

Because previous reports did not describe what types of Ca²⁺ channels are activated by NA in CHO-_1A, CHO-_1B, and CHO-_1D, we attempted to characterize the Ca²⁺ channels activated by NA in these three cell types using whole-cell patch clamp and [Ca²⁺]_i monitoring. In CHO cells, VOCCs do not seem to be involved in the NA-induced increase in [Ca²⁺]_i for the following reasons: 1) CHO cells are nonexcitable cells that usually lack VOCCs (Perez et al., 1993); and 2) the NA-induced increase in [Ca²⁺]_i was resistant to specific blockers of L-type VOCC such as nifedipine (data not shown). The whole-cell currents induced by NA in CHO-_1A, CHO-_1B, and CHO-_1D are conducted through Ca²⁺-permeable NSCCs for the following reasons: 1) the current-voltage relationships were linear and their reversal potentials were close to 0 mV (Fig. 4), indicating that the current is conducted through either NSCCs or Cl channels; 2) the reversal potential of the NA-induced currents was not affected by a change in the concentration of Cl in the bath solution (see Results), indicating that the current is carried through NSCCs; and 3) the NSCC is permeable to Ca²⁺, because a current could be induced in a bath solution containing only Ca²⁺ as the movable cation (see Results). Based on their pharmacology (sensitive to LOE 908 and resistant to SK&F 96365) (Fig. 3), these channels are different from SOCCs (which are sensitive to SK&F 96365 and resistant to LOE 908) (Fig. 6). The result that thapsigargin induced sustained increase in [Ca²⁺]_i in CHO-_1A pretreated with 100 nM NA (Fig. 6C) may be supported this indication. Moreover, NA may activate the same Ca²⁺ channels (NSCCs) in CHO-_1A, CHO-_1B, and CHO-_1D (Figs. 3 and 4).

Next, we investigated whether the Ca²⁺ influx through NSCCs plays a role in the NA-induced arachidonic acid release. As reported previously using a number of cell types (Apkon and Nerbonne, 1988; Weiss and Insel, 1991), NA stimulated arachidonic acid release in CHO-_1A, CHO-_1B, and CHO-_1D (Fig. 7). The degree of the NA-induced increase in arachidonic acid release in CHO-_1A, CHO-_1B, and CHO-_1D was similar (Fig. 7). The NA-induced increase in arachidonic acid release requires the influx of extracellular Ca²⁺, because this response was blocked by EGTA (Fig. 7). Moreover, the NA-induced increase in arachidonic acid release was inhibited by pertussis toxin (data not shown). A previous study reported that ₁-ARs can couple directly to phospholipase A₂ activation via a pertussis toxin-sensitive pathway and then activate arachidonic acid release (Perez et al., 1993). The previous report was based on the finding that stimulation of arachidonic acid release was not affected even when both ₁-AR-stimulated polyphosphoinositide hydrolysis (PI) and the increase in [Ca²⁺]_i were blocked by neomycin. However, the activation of NSCCs by NA is independent of the stimulation of PI, because the EC₅₀ value of NA for activating NSCCs (1-10 nM) was less than that for stimulating IPs accumulation (10-100 nM) (Figs. 2 and 5). Moreover, LOE 908 at a concentration of 3 µM, which inhibited the NA-induced sustained increase in [Ca²⁺]_i (Fig. 3), blocked the NA-induced increase in arachidonic acid release (Fig. 7). These results suggest that, in addition to pertussis toxin-sensitive pathway, the Ca²⁺ influx through NSCCs may play an important role in the NA-induced enhancement of arachidonic acid release in CHO-_1A, CHO-_1B, and CHO-_1D (Fig. 8).

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Schematic representation of signaling pathways for arachidonic acid release activated by NA in CHO cells stably expressing 1-ARs. NA activates LOE 908-sensitive and SK&F 96365-resistant NSCCs. Both PTX-sensitive cascade and extracellular Ca2+ influx through NSCC are indispensable for NA-induced arachidonic acid release in CHO cells stably expressing 1-ARs. See text for details.

In summary, the LOE 908-sensitive Ca²⁺-permeable NSCC is activated by NA in CHO-_1A, CHO-_1B, and CHO-_1D, and extracellular Ca²⁺ influx through NSCC plays an important role in the NA-induced increase in arachidonic acid release in these three cell types.